This proposal is to conduct a systematic investigation of the kinetics of disposition of cyclophosphamide (CP), an antitumor agent used by seven million patients annually, and its key alkylating metabolites, 4-hydroxy cyclophosphamide/aldophosphamide (4-OH CP/AP), phosphoramide mustard (PM), and nor-nitrogen mustard (HN2) in the rat. The plasma concentration-time profile will be measured following individual administration of each synthetically prepared drug and metabolite and relevant pharmacokinetic parameters such as half-lives, clearance, and volumes of distribution will be assessed. We propose to measure these plasma concentrations using a gas chromatographic-mass spectrometric method for PM and HN2 newly developed in our laboratory. In the case of CP and 4-OH CP/AP, we will modify literature GC-MS methodology. Additionally, the kinetics of sequential metabolism of CP and metabolites will be studied using the newly developed principles of metabolite kinetics of Pang and Gillette, assuming that the liver is the sole eliminating organ. Unlabeled precursor and stable isotopically labeled metabolite will be simultaneously administered into intravenous and intraportal routes, respectively, of the rat. Quantitation of the metabolites derived from each route will be performed using a second stable isotopically labeled derivative of the relevant metabolite. The hepatic extraction ratios, clearances, fractional rate conversions of CP to the individual alkylating metabolites, the availabilities of these metabolites for sequential metabolism, the rate constants of metabolite formation will be estimated. Additionally, the absolute bioavailability of CP in the rabbit and in eight cancer patients will be estimated using simultaneous oral and i.v. administrations of deuterium labeled and unlabeled CP, respectively and similar GC-MS analytical method. These studies represent the first in vivo application of the priniples of metabolite kinetics of an important anticancer drug. In addition, the kinetics of CP disposition enables us to examine the extension of these principles from to sequential steps to a multiple step kinetic pathways. These studies will also provide an in depth understanding of the pharmacokinetics and pharmacology of CP and give an insight to the potential metabolite interaction of CP with other agents.